According to Beer's Law, absorbance increases as the pathlength or thickness of a sample increases. Many samples for which the analytical technique of preference is infrared spectroscopy, such as polymers, solvents and lubricants, are inherently highly absorbing. It is difficult to obtain useful information about these samples by spectroscopic analysis if the apices of the absorbance peaks generated by the spectrophotometer of are too high, i.e. the absorbance peaks will tend to exceed the "Y" axis or ordinate scale of the spectrophotometer. When the apex of an absorbance peak exceeds the "Y" axis of the analyzer, it is impossible to determine the intensity of the peak with acceptable accuracy. In addition, the absorbance peaks of such samples tend to obscure each other if the absorbance peaks are too intense, as is often the case with relatively thick samples.
Accordingly, it is difficult to compare the spectra of samples that have obscured absorbance peaks with spectra of other samples that do not have the same peaks obscured. It is also difficult to compare spectra with peaks that are obscured by other more intense peaks with spectra of samples made from films that show more absorbance peaks. In addition, the optical materials between which samples are deposited produce interference fringes and can absorb energy both of which can obscure absorbance peaks. Thus, the spectrometric analysis of films is rendered problematical in part because the absorbance measurements can be obscured causing difficulty in reading and comparing absorbance peaks.
It is known that more readable absorbance patterns can be obtained if the thickness of the film is reduced. There are techniques available for producing samples from liquids and viscous materials. These include transmission sampling using liquid cells, pressing thin films from polymers using polished platens and laboratory presses, and internal reflection and specular reflection spectroscopy. All of these techniques have certain limitations in providing suitable samples for analysis especially spectroscopic analysis.
Transmission sampling using cells requires that the sample be injected into the cell with a syringe or under pressure, which makes filling of the cell somewhat cumbersome. The technique is not practical for highly viscous samples and for samples which adversely affect the cell such as adhesives. Using presses and dies is cumbersome and is not practical for liquids. Internal reflection and specular sampling produces spectra which are not directly comparable with transmission spectra and conversion of the results is cumbersome and error prone. The internal reflection technique cannot be used to produce thin films as there is no practical means of accurately controlling sample thickness, and the optics obscure the detail of spectra in the 600 to 400 cm-1 range.
In other fields, such as cytology, thin samples are desirable because if the specimen comprises several layers of cells (a relatively thick sample) it is difficult to distinguish one layer from another when one layer of cells is overlaid with another layer of cells. Thin films are also useful for testing the physical properties of samples, such as tensile strength testing.
Liquid samples have traditionally been tested in spectrophotometers by placing the sample in a vessel also known as a cell. Cells are comprised of sealed cavities bounded by a pair of windows made from optical materials which, when mounted in the beam of energy (e.g. light) emitted by the energy source of the spectrophotometer, will not absorb the energy that passes through them. The cells are typically mounted vertically in the sample compartment of the spectrophotometer. Liquid cells used in infrared spectrophotometers are normally filled with a syringe, as cell volumes and pathlengths (the space between the windows) tend to be small.
Another spectroscopic sampling technique is known as the attenuated total reflectance (ATR) sampling technique (also known as multiple internal reflection (MIR) sampling). In this technique, the sample is placed against the planar surface of a prism made from an optical material with a high refractive index, and the beam of energy of the spectrophotometer is directed through the end of the prism which is at an angle (usually 30 to 60 degrees). The beam bounces one or more times against the sample.
This technique was originally practiced with accessories used in the vertical position, but it was impractical to use it with liquids because there was no means of containing the liquid sample in a manner that would keep the sample in contact with the face of the prism. In about 1980, an improvement to the ATR technique was introduced to enable analysis of the sample in the horizontal position. This innovation allowed the use of ATR accessories with liquids and it has become popular because the sample can simply be poured into the accessory and then dumped out after the sample has been tested in a spectrophotometer.
ATR techniques require an optical bench comprised of mirrors to direct the beam from the energy source through the prism and then to the detector of the instrument, whereas a cell sits directly in the beam of the instrument and the spectrophotometer beam passes in a straight line from the energy source through the sample and on to the detector of the instrument. Both ATR spectroscopy and transmission spectroscopy using a cell require the use of some optical material as a substrate (the prism) or to hold the sample (transmission windows).
It would be an advance in the art of producing samples for analysis especially by infrared spectroscopy to be able to produce films that are relatively thin from highly absorbing samples that can be analyzed in the transmission mode without the need for cumbersome techniques such as liquid cells and polymer film pressing and which eliminate some of the inherent shortcomings of simpler techniques such as internal reflection sampling. It would be an advance in the art of producing tissue and cell samples to be able to produce controlled thickness thin films which approach the thickness of a single layer of cells.